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A high molecular mass cranberry constituent reduces mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite

Ervin I. Weiss , Avital Kozlovsky , Doron Steinberg , Ron Lev-Dor , Ronit Bar Ness Greenstein , Mark Feldman , Nathan Sharon , Itzhak Ofek
DOI: http://dx.doi.org/10.1016/S0378-1097(04)00035-7 89-92 First published online: 1 March 2004

Abstract

Previous investigations showed that a high molecular mass, non-dialyzable material (NDM) from cranberries inhibits the adhesion of a number of bacterial species and prevents the co-aggregation of many oral bacterial pairs. In the present study we determined the effect of mouthwash supplemented with NDM on oral hygiene. Following 6 weeks of daily usage of cranberry-containing mouthwash by an experimental group (n=29), we found that salivary mutans streptococci count as well as the total bacterial count were reduced significantly (ANOVA, P<0.01) compared with those of the control (n=30) using placebo mouthwash. No change in the plaque and gingival indices was observed. In vitro, the cranberry constituent inhibited the adhesion of Streptococcus sobrinus to saliva-coated hydroxyapatite. The data suggest that the ability to reduce mutans streptococci counts in vivo is due to the anti-adhesion activity of the cranberry constituent.

Keywords
  • Cranberry
  • Mutans streptococcus
  • Saliva
  • Mouthwash
  • Anti-adhesion
  • Antibacterial
  • Hydroxyapatite

1 Introduction

Indications of possible means other than antibacterial therapy for controlling infectious diseases have focused research on bacterial adhesion mechanisms. Attempts to apply the knowledge thus obtained to the development of anti-adhesion therapy for such diseases have been made [1].

Previous investigations showed that a high molecular mass, non-dialyzable material (NDM) obtained from cranberries strongly inhibits the adhesion of a number of bacterial species [2,3] and prevents the co-aggregation of many oral bacterial pairs [4]. NDM did not exhibit bactericidal effects against a variety of Gram-negative and Gram-positive oral bacteria tested [5], consistent with previous studies showing that cranberry juice lacks bactericidal activity [6]. The anti-adhesive properties of whole cranberry juice either as pure fruit juice or in a mouthwash formulation cannot be tested in the oral cavity because cranberry juice contains a high level of sucrose and is acidic. The high sugar content promotes growth of dental plaque bacteria and is considered a major cariogenic factor whereas the acidity of the juice may cause tooth decalcification.

Nevertheless, cranberry constituents exhibiting anti-adhesion activity and devoid of carries-promoting agents would be expected to control the oral flora. In the present study we examined the effect of a NDM-containing mouthwash on salivary bacterial counts in healthy volunteers.

2 Materials and methods

2.1 NDM mouthwash

NDM was isolated from cranberry juice concentrate (Ocean Spray, Inc.) as described [7]. Briefly, NDM was obtained after lyophilization of the material retained in a dialysis bag (12 000 molecular mass cut-off) following extensive dialysis of the juice. It exhibits tannin-like properties and it is soluble in water, devoid of proteins and fats, and contains 56.6% carbon and 4.14% hydrogen [8]. NDM was incorporated at a final concentration of 3 mg ml−1 into a standard mouthwash containing sorbitol (5%), sodium benzoate (0.1%), sodium fluoride (0.05%), alcohol (7.0%), menthol (0.05%), eucalyptus oil (0.1%), FD&C Blue 1 (0.0001%), and PEG-20 glyceryl isostearate (0.6%) in distilled water.

2.2 Pilot study

In the pilot study, three healthy volunteers were asked to rinse their mouths with 15 ml of saline to establish the baseline bacterial count. Immediately afterwards, they rinsed twice with 15 ml of NDM mouthwash for 30 s, at a 1-min interval, followed by a 5-ml saline rinse. Samples of the saline expectorates were plated on tryptic soy blood agar plates for total bacterial counts and on mitis salivarius agar plates supplemented with bacitracin for mutans streptococci counts. The volunteers continued to use the NDM mouthwash as above twice daily for 2 weeks. During this time they were instructed to refrain from using any other mouthwash but to continue their regular oral hygiene habits. Samples of saline expectorates were obtained on day 14 and plated as above.

2.3 Clinical trial

A total of 60 healthy volunteers (24 males and 36 females, average age 26, range 21–39 years) from the student population of Tel Aviv University were included a two-cell, double-blind, clinical trial. Exclusion criteria included antibiotic treatment within 1 month prior to the study, oral soft tissue pathology other than marginal gingivitis, missing teeth, untreated visible carious lesions and smoking. The subjects were randomly assigned to the experimental group (n=30) or to the placebo group (n=30), which used the same mouthwash preparation but without NDM. All volunteers were asked to sign their informed consent, according to the protocol of the Human Subject Experiment Committee of the Tel Aviv University Medical School.

A two-cell, double-blind study was carried out on volunteers using the NDM mouthwash twice daily for 42 days. The daily regimen was as that in the pilot study. To ensure compliance, the volunteers were asked to keep to their regular hygiene habits during the 42-day study. On day 0, clinical and microbial measurements (described below) were performed at least 8 h after the last oral hygiene procedure and the subjects were instructed to refrain from eating and drinking 3 h before measurements were taken. On day 42 of the study, measurements were made as on day 0. Twenty-nine of the experimental group subjects and all 30 placebo group subjects completed the study.

2.4 Clinical and microbial measurements

The following measurements were made on days 0 (baseline) and 42 of the study: (i) plaque index – plaque accumulation was evaluated on the buccal surfaces following the application of disclosing solution (Butler GUM, Butler, Chicago, IL, USA), according to the Turesky modification of the Quigley–Hein plaque index [9]; (ii) gingival index – gingival inflammation was assessed on the buccal and lingual marginal gingivae and the interdental papillae, according to the Loe–Silness gingivitis index [10]; and (iii) salivary bacteria were enumerated by plating a sample of unstimulated whole saliva on a DiaSlide (Savyon Diagnostics, Ashdod, Israel), a culturing device [7] modified to test oral bacterial samples using trypticase soy blood agar for total bacterial counts and mitis salivarius agar for mutans streptococci counts. The bacteria were sampled and cultured according to the manufacturer's instructions.

Comparisons were made using analysis of variance (ANOVA) and treatment effects were compared using the paired t-test with Bonferroni correction.

2.5 The effect of NDM on Streptococcus sobrinus adhesion in vitro

Adhesion of S. sobrinus to saliva-coated hydroxyapatite was determined as described previously [11,12]. Briefly, unstimulated whole saliva samples were collected from a single donor and clarified by centrifugation. One ml of saliva diluted 1:1 with phosphate-buffered saline (PBS) was added to 40 mg hydroxyapatite beads (Bio-Rad Laboratories, Hercules, CA, USA) and incubated with gentle agitation at 37°C for 1 h. Following incubation, the beads were washed with PBS to remove unbound saliva. Radiolabeled S. sobrinus 6715 were prepared by growing the bacteria to the late exponential phase in BHI medium supplemented with 5 μCi of [3H-methyl]thymidine (NEN Products, Boston, MA, USA) per ml BHI, at 37°C under aerobic conditions in the presence of 5% CO2. 3H-labeled bacteria were incubated with NDM at a final concentration of 0, 66, 83, 130, 260, and 1330 μg ml−1 in PBS containing 4% sucrose, for 1 h at 37°C. The beads were then washed free of unbound bacteria, rinsed with 4 ml PBS and the bacteria-associated radioactivity of the beads was counted (Beta-matic, Kontron, Switzerland).

3 Results and discussion

In view of the finding that NDM strongly inhibits the adhesion of a number of bacterial species [2,3], we tested its effect on salivary bacteria in humans. Measuring salivary bacterial counts is one of the most accepted in vivo methods for evaluation of mouthwash activity and is considered a useful predictor of active ingredient substantivity [13] and the persistence of its effect [1416]. In the pilot study three healthy volunteers rinsed their mouths with NDM-containing mouthwash. Total bacteria and mutans streptococci counts in the saline samples before and immediately after using NDM did not differ significantly. However, after rinsing their mouths for 14 days, there was a significant decrease in both total bacteria (1.2×107±0.6 on day 14 versus 3.5×107±0.8 on day 0, P<0.05), and in mutans streptococci (0.3×105±0.6 on day 14 versus 3.8×105±0.7 on day 0, P<0.01). Encouraged by these preliminary results, we embarked on a two-cell, double-blind clinical trial in which 60 volunteers were included. There was no difference in bacterial counts between the experimental group and the placebo group on day 0. The bacterial counts in the whole saliva of volunteers using NDM mouthwash showed a marked reduction in both mutans streptococci and total bacteria between day 0 and day 42 of the study as compared with that in the placebo mouthwash group (ANOVA, P<0.01) (Fig. 1). Furthermore, a comparison on day 42 between the group using the NDM mouthwash and the group using the placebo mouthwash also showed a marked difference in bacterial count. The results of the gingival and plaque indices showed no significant differences between the two groups and between day 0 and day 42 of the study (data not shown), in contrast to mouthwashes containing potent antibacterial agents such as chlorhexidine which significantly affects these indices. This may be due to differences in the mechanism of action between anti-adhesion and bactericidal agents, where the magnitude of reduction in bacterial counts is immediate and more pronounced by the latter. Thus, the present clinical trial does not suggest that NDM-containing mouthwashes have a clinical advantage.

1

Effect of NDM mouthwash on total bacterial counts (A) and mutans streptococci counts (B) in unstimulated whole saliva. Hatched bar=group rinsing with NDM mouthwash (n=29), gray bar=placebo group (n=30). The values are the mean±S.D. of log colony forming units. Treatment effect and time effect were significant (ANOVA, P<0.01) for both mutans streptococci and total bacterial counts.

We next studied the effect of NDM on S. sobrinus adhesion to saliva-coated hydroxyapatite. NDM inhibited the sucrose-dependent adhesion of S. sobrinus to saliva-coated hydroxyapatite in a dose-dependent manner. Maximal inhibition (90%) was observed at 130 μg ml−1 of NDM (Fig. 2).

2

Effect of NDM on sucrose-dependent adhesion of mutans streptococci to saliva-coated hydroxyapatite. 3H-labeled S. sobrinus 6715 was added to saliva-coated hydroxyapatite beads in the presence of the indicated concentrations of NDM. After 1 h incubation at 37°C, the beads were washed free of non-adherent streptococci and the associated radioactivity was determined. The values are the mean±S.D. of triplicate experiments.

Because NDM was shown to have no effect on the viability of mutans streptococci, including S. sobrinus (not shown), or on the viability of other plaque bacteria [5], we reasoned that the reduction in mutans streptococci counts in our study was due to the anti-adhesion effect of this compound. The concept of using anti-adhesion agents to reduce bacterial colonization was demonstrated by Kelly et al. [17]. They showed in four volunteers that a synthetic peptide adhesin analog, which inhibits the in vitro adhesion of S. mutans, reduced tooth colonization and lowered S. mutans level in saliva.

Cumulatively, these studies support the hypothesis that anti-adhesion agents can alter the oral flora. This concept is particularly relevant to the NDM derived from cranberries, which is edible and non-fermentable, whereas other known adhesion inhibitors of oral bacteria, such as galactose and lactose, are fermentable and hence cariogenic or promote plaque development.

Continuous use of mouthwashes containing safe anti-adhesion agents may represent a novel approach to altering biofilm formation on teeth. Their potential in controlling dental caries merits further investigation.

Acknowledgements

This study was performed in part at the R. Goldstein Research Center, Faculty of Dentistry, Hebrew University-Hadassah, and at the Alpha Omega Research Laboratories, School of Dental Medicine, Tel Aviv University. The work was supported in part by a grant from Ocean Spray, Inc.

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